Malignant canine mammary epithelial cells shed exosomes containing differentially expressed microRNA that regulate oncogenic networks

Abstract Background Breast (mammary) cancers in human (BC) and canine (CMT) patients share clinical, pathological, and molecular similarities that suggest dogs may be a useful translational model. Many cancers, including BC, shed exosomes that contain microRNAs (miRs) into the microenvironment and circulation, and these may represent biomarkers of metastasis and tumor phenotype. Methods Three normal canine mammary epithelial cell (CMEC) cultures and 5 CMT cell lines were grown in serum-free media. Exosomes were isolated from culture media by ultracentrifugation then profiled by transmission electron microscopy, dynamic light scattering, and Western blot. Exosomal small RNA was deep-sequenced on an Illumina HiSeq2500 sequencer and validated by qRT-PCR. In silico bioinformatic analysis was carried out to determine microRNA gene and pathway targets. Results CMEC and CMT cell lines shed round, “cup-shaped” exosomes approximately 150–200 nm, and were immunopositive for exosomal marker CD9. Deep-sequencing averaged ~ 15 million reads/sample. Three hundred thirty-eight unique miRs were detected, with 145 having > ±1.5-fold difference between one or more CMT and CMEC samples. Gene ontology analysis revealed that the upregulated miRs in this exosomal population regulate a number of relevant oncogenic networks. Several miRNAs including miR-18a, miR-19a and miR-181a were predicted in silico to target the canine estrogen receptor (ESR1α). Conclusions CMEC and CMT cells shed exosomes in vitro that contain differentially expressed miRs. CMT exosomal RNA expresses a limited number of miRs that are up-regulated relative to CMEC, and these are predicted to target biologically relevant hormone receptors and oncogenic pathways. These results may inform future studies of circulating exosomes and the utility of miRs as biomarkers of breast cancer in women and dogs

Cytokine Gene Expression in Response to SnSAG1 in Horses with Equine Protozoal Myeloencephalitis

Equine protozoal myeloencephalitis (EPM) is a neurologic syndrome seen in horses from the Americas and is mainly caused by Sarcocystis neurona. Recently, a 29-kDa surface antigen from S. neurona merozoites was identified as being highly immunodominant on a Western blot. This antigen has been sequenced and cloned, and the expressed protein has been named SnSAG1. In a previous study, cell-mediated immune responses to SnSAG1 were shown to be statistically significantly reduced in horses with EPM in comparison to EPM-negative control horses. It therefore appears as though the parasite is able to induce immunosuppression towards parasite-derived antigens as parasite-specific responses are decreased. Isolated peripheral blood lymphocytes from 21 EPM (cerebrospinal fluid [CSF] Western blot)-negative horses with no clinical signs and 21 horses with clinical signs of EPM (CSF Western blot positive) were cocultured with SnSAG1 for 48 and 72 h, and the effect on cytokine production was investigated by means of reverse transcriptase PCR. Cytokines assayed include gamma interferon (IFN-γ), tumor necrosis factor alpha, interleukin (IL)-2, IL-4, and IL-6. β-Actin was used as the housekeeping gene. A Wilcoxon signed-rank test of the findings indicated that there was a statistically significant decrease in IFN-γ production after 48 h in culture for samples from horses with clinical disease. There was also a statistically significant increase in IL-4 production after 72 h in culture for samples from horses with EPM. These results further support the notion that this parasite is able to subvert the immune system in horses with clinical disease